High-risk human papillomavirus (HPV) infection is the primary risk factor for cervical cancer, which is the second most prevalent cancer in women worldwide causing 15% of female cancer mortality. Papillomaviruses establish persistent infection by maintaining their genomes as episomes in infected cells. In the HPV life cycle that is tightly linked to the differentiation program of host epithelium, E2 ensures that the viral genome is established, replicated and maintained in the early-infected basal epithelial cells. During epithelium differentiation, E2 also contributes to the tight regulation of the viral oncogene transcription to create a conducive environment for successful completion of the viral life cycle. Loss of E2 expression leads to dysregulated viral oncogene expression and has been mechanistically linked to malignant progression of HPV positive lesions. Our previous work identified the cellular protein Brd4 (bromodomain-containing protein 4) as a novel receptor for E2. Brd4-E2 interaction functions in viral episome maintenance, viral transcriptional activation and repression of the viral oncogenes. Our studies established that Brd4 is highly expressed in the basal epithelial layer, supporting its role in E2 functions during the early phase of the viral life cycle. However, little is known about how Brd4 regulates the multiple functions of E2, nor is it clear how this virus-host interaction contributes to the differentiation- dependent HPV life cycle. This grant application aims to identify and characterize additional cellular components that regulate the E2-Brd4 functions, to determine how Brd4 contributes to E2 transcriptional regulation, and to investigate the functional impact of E2-Brd4 interactions in the HPV life cycle during epithelium differentiation. Infection by the HPV is the most common sexually transmitted agent, afflicting 50-80% of the population. This research will provide greater understanding of the molecular mechanisms that regulate the HPV life cycle and malignant progression. The new mechanisms identified in this study will provide a point of departure for developing new compounds to abrogate the virus-host interaction and cure HPV persistent infections. Mechanistic insights into how E2-Brd4 interactions repress the viral oncogenes that account for the tumorigenic nature of HPV-associated diseases will offer promising leads for novel therapeutic strategies. This study will provide a paradigm for other episomal DNA tumor viruses, including Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, both of which also target Brd4. PUBLIC HEALTH RELEVANCE: High-risk human papillomaviruses (HPVs) are strongly associated with cervical cancer, which is the second most common cancer in women worldwide causing 15% of female cancer mortality. This proposal will investigate the molecular mechanisms that regulate the papillomavirus and host interactions during the HPV life cycle and malignant progression. Our study will identify novel targets for developing therapeutic approaches to cure HPV persistent infection and associated human cancers.